Mutant rho factors with increased transcription termination activities. II. Identification and functional dissection of amino acid changes.

We have determined the nucleotide sequences of three mutant rho genes encoding hyperfunctional rho proteins (rho S) together with their parent allele, rho-ts702. These mutant rho factors contain the following amino acid changes as deduced from their sequences: (1) the thermo-labile mutant, rho-ts702, has Thr304 substituting for Ala; (2) rho S-77 and rho S-81, which are selectively altered in the primary polynucleotide binding site, share an identical mutation, Leu3----Phe; (3) rho S-82, which is altered in both the primary and secondary polynucleotide binding sites, carries three amino acid substitutions together, Leu3----Phe, Asp156----Asn and Thr323----Ile. Dissection and functional characterization of each mutation in rho S-82 have revealed that Ile323 alone is responsible for alterations in both the secondary RNA interaction and the terminator selectivity observed with the original mutant, rho S-82. Taken together, these results not only confirm our proposal in the accompanying paper that the primary and secondary RNA binding sites differently contribute in determining the overall efficiency and site-specificity of termination, respectively, but also support the possibility that these binding sites exist as structurally distinct domains in rho protein. In contrast, Asn156 was shown to cause decreased termination efficiency, though it had no influence on RNA interactions. Thus, this amino acid residue appears to be associated with still another rate-determining step of termination, for instance, interactions between rho and RNA polymerase. On the basis of Chou-Fasman secondary structure predictions as well as amino acid sequence comparison with F1-ATPase, we discuss how the proposed domains are structurally and functionally related to the putative ATPase reactive center of rho protein.